Combination therapy

ABSTRACT

Pharmaceutical compositions and treatments involving iNKT cell activation are provided.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.provisional application Ser. No. 61/900,186, filed Nov. 5, 2013, thecontent of which is incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

Modulating the immune system has been pursued as a desirable approach totreat a variety of diseases and disorders, including, but not limitedto, autoimmune disease, infection, allergy, inflammatory conditions,spontaneous abortion, pregnancy, graft versus host disease and cancer. Tcells have been a target of such modulation. T cells are lymphocytesthat participate in multiple immune system functions. Subsets of T cellssuch as helper T cells, cytotoxic T cells and suppresser T cells,mediate different immunologic functions. Natural killer T (NKT) cellsare a subset of T lymphocytes that share surface markers and functionalcharacteristics with both conventional T cells and natural killer (NK)cells. Unlike T cells, they recognize glycolipid antigens rather thanpeptide antigens.

NKT cells can be divided into three subsets: Type 1 which express aninvariant T cell receptor and are CD1d-restricted (iNKT), Type 2 (NKT)which express varied T cell receptors, but are CD1d-restricted, and Type3 which do not express the invariant T cell receptor and are notCD1d-restricted (NKT-like). Type 1 iNKT cells express a uniquelyrearranged, highly conserved, semi-invariant TCR-α chain (Vα24-Jα18 inhumans and Vα14-Jα18 in mice), which preferentially pairs with specificTCR-β chains (Vβ11 in humans or Vβ8.2, Vβ7 and Vβ2 in mice). They arehighly conserved throughout animal phylogeny. This is in contrast tomost T cell subpopulations, which have diverse sequences for their TCell Receptors (TCRs). The invariant TCR of iNKT cells recognizesglycolipid antigens presented on the MHC-I-like protein CD1d on thesurface of antigen presenting cells. A hallmark of iNKT cells is theircapacity to rapidly produce a mixture of cytokines, including IFNγ andIL-4, which are signature cytokines otherwise produced by T helper typeI (Th1) and Th2 cells, respectively. Invariant NKT cells are sometimesreferred to as “Classical NKT Cells”.

A unique feature of iNKT cells is that they recognize and are activatedby the marine sponge-derived glycolipid, α-Galactosyl-Ceramide(α-GalCer), presented on CD1d. This has been utilized, for example, tomonitor iNKT cells by flow cytometry, by using α-GalCer-loaded CD1dtetramers. The mouse monoclonal antibody 6B11, which binds to theinvariant loop of the human-iTCR, has also been used to monitor humanand NHP iNKT cells. The pathways associated with iNKT cell activation byα-GalCer also have been studied.

iNKT cells develop in the thymus, similar to other T cells. Studies inmice show that iNKT cells, unlike conventional T cells, acquired amemory phenotype during their natural development by recognizinghitherto unknown, endogenous antigens presented on CD1d molecules, andwithout requiring prior exposure to foreign or pathogenic antigens. Dueto their memory phenotype, they can be rapidly activated and expandwithin the peripheral immune compartment in response to exposure toforeign or endogenous glycolipid antigens presented byantigen-presenting cells (APCs).

iNKT cells share characteristics of both the innate and adaptive arms ofthe immune system and thus play a unique role by modulating T and B cellresponses as well as innate immunity. iNKT cells are rapid-onset whichis a feature of the innate immune system. They also display features ofthe adaptive immune system because they share properties of other Tcells such as antigen specific responses. As such, they serve as abridge between the two systems where they can play both pro-inflammatoryand immuno-regulatory roles either to enhance or attenuate developingimmune responses, respectively.

The properties of iNKT cells has prompted investigations into themanipulation of iNKT cell function as a treatment for disease. Numerousstudies have shown that iNKT cells can regulate the balance between Th1and Th2 responses. These cells are postulated to play a role in theresponse to pathogens, in immune surveillance in cancer, and in theregulation of autoimmunity. For most of these conditions, the iNKT celldefect has only been partially characterized and in some cases has beendisputed by contradictory studies. Human studies, in particular, areconstrained by two important limitations. First, most human studies haveused suboptimal methods for the identification of iNKT cells. Second,most human studies are qualitative only, and little human data existsrespecting the functional consequences of modulation of iNKT cellnumbers, ratios, or function.

Studies to date have used an indirect approach to iNKT cell stimulationvia the presentation of the activating glycolipid ligand alphagalactosylceramide or other stimulatory glycolipids by dendritic cellsor other antigen presenting cells (APCs) that express the MHC class Ilike molecule CD1d. What is not known is if direct activation of iNKTcells by use of a direct iNKT binding antibody without the presentationof a glycolipid or activating cytokines via APC/iNKT cell binding canhave a similar effect.

The PCT published application WO 2013/063395 discloses for the firsttime humanized antibodies that bind human iNKT cells, includingantibodies that can activate iNKT cells in vivo and antibodies that candeplete iNKT cells in vivo. This application provides for the first timethe opportunity to test such antibodies in human systems as therapeuticsfor the treatment of disease.

SUMMARY OF THE INVENTION

It has been discovered that combination treatment with both an antibodythat activates iNKT cells and a Programmed Death (PD-1) antagonist issurprisingly effective in enhancing an immune response in the treatmentof an established cancer. It is also believed that these findings extendto infectious disease. The treatment is effective even without separateadministration of an isolated antigen.

It also has been demonstrated that direct activation of iNKT cells canadequately stimulate iNKT cells to provide both prophylactic andtherapeutic anti-tumor activity when administered. These results suggesta new approach to the activation of iNKT cells for the treatment ofcancer.

According to one aspect of the invention, a method is provided fortreating a human subject having a cancer or an infection. The methodcomprises administering to the human subject an effective amount of (a)an isolated humanized antibody that selectively binds and activates iNKTcells, and (b) an isolated Programmed Death (PD-1) antagonist, whereinthe isolated humanized antibody and the isolated Programmed Death (PD-1)antagonist are administered in amounts effective to treat the cancer orthe infection. In some embodiments, the treatment is without concurrentadministration of an antigen containing vaccine. In some embodiments,the treatment further comprises administering an antigen containingvaccine.

The cancer can be, for example, melanoma, squamous cell carcinoma, basalcell carcinoma, breast cancer, head and neck carcinoma, thyroidcarcinoma, soft tissue sarcoma, bone sarcoma, testicular cancer,prostatic cancer, ovarian cancer, bladder cancer, skin cancer, braincancer, angiosarcoma, hemangiosarcoma, mast cell tumor, primary hepaticcancer, lung cancer, pancreatic cancer, gastrointestinal cancer, renalcell carcinoma, hematopoietic neoplasia, or a metastatic cancer thereof.In some embodiments, the cancer is melanoma. In some embodiments, thecancer is renal cell carcinoma.

According to another aspect of the invention, a pharmaceuticalcomposition is provided. The pharmaceutical composition contains aneffective amount of (a) an isolated humanized antibody that selectivelybinds and activates iNKT cells, and (b) an isolated Programmed Death(PD-1) antagonist. In any of the embodiments, the pharmaceuticalcomposition is sterile. In any of the embodiments, the pharmaceuticalcomposition can further include a pharmaceutically acceptable carrier.

According to another aspect of the invention, a kit is provided. The kitincludes a package containing (a) an isolated humanized antibody thatselectively binds and activates iNKT cells, and (b) an isolatedProgrammed Death (PD-1) antagonist. In some embodiments, the kit doesnot include an antigen containing vaccine. In some embodiments, the kitincludes an antigen containing vaccine.

In any of the embodiments, the isolated Programmed Death (PD-1)antagonist can be a peptide that binds PD-1, a humanized antibody thatselectively binds PD-1, a humanized antibody that selectively bindsPD-L1, a humanized antibody that selectively binds PD-L2, or acombination thereof. In any of the embodiments, the PD-1 antagonist canbe any of the specific such PD-1 antagonists described in greater detailbelow. In any of the embodiments, the isolated humanized antibody thatselectively binds and activates iNKT cells can be any of the humanizedantibodies that selectively bind and activate iNKT cells described ingreater detail below. In any of the embodiments, the isolated humanizedantibody that selectively binds and activates iNKT cells can be NKTT320.

It further has been discovered that combination treatment with both anantibody that activates iNKT cells and a CTLA-4 antagonist issurprisingly effective in enhancing a immune response in the treatmentof an established cancer. It is also believed that these findings extendto infectious disease. The treatment is effective even without separateadministration of an isolated antigen.

According to one aspect of the invention, a method is provided fortreating a human subject having a cancer or an infection. The methodcomprises administering to the human subject an effective amount of (a)an isolated humanized antibody that selectively binds and activates iNKTcells, and (b) an isolated CTLA-4 antagonist, wherein the isolatedhumanized antibody and the isolated CTLA-4 antagonist are administeredin amounts effective to treat the cancer or the infection. In someembodiments, the treatment is without concurrent administration of anantigen containing vaccine. In some embodiments, the treatment furthercomprises administering an antigen containing vaccine.

The cancer can be, for example, melanoma, squamous cell carcinoma, basalcell carcinoma, breast cancer, head and neck carcinoma, thyroidcarcinoma, soft tissue sarcoma, bone sarcoma, testicular cancer,prostatic cancer, ovarian cancer, bladder cancer, skin cancer, braincancer, angiosarcoma, hemangiosarcoma, mast cell tumor, primary hepaticcancer, lung cancer, pancreatic cancer, gastrointestinal cancer, renalcell carcinoma, hematopoietic neoplasia, or a metastatic cancer thereof.In some embodiments, the cancer is melanoma. In some embodiments, thecancer is renal cell carcinoma.

According to another aspect of the invention, a pharmaceuticalcomposition is provided. The pharmaceutical composition contains aneffective amount of (a) an isolated humanized antibody that selectivelybinds and activates iNKT cells, and (b) an isolated CTLA-4 antagonist.In any of the embodiments, the pharmaceutical composition can be sterileis sterile. In any of the embodiments, the pharmaceutical compositioncan further include a pharmaceutically acceptable carrier.

According to another aspect of the invention, a kit is provided. The kitincludes a package containing (a) an isolated humanized antibody thatselectively binds and activates iNKT cells, and (b) an isolated CTLA-4antagonist. In some embodiments, the kit does not include an antigencontaining vaccine. In some embodiments, the kit includes and antigencontaining vaccine.

In any of the embodiments, the isolated CTLA-4 antagonist can be apeptide that binds CTLA-4, a humanized antibody that selectively bindsCTLA-4, a humanized antibody that selectively binds a ligand of CTLA-4,or a combination thereof. In any of the embodiments, CTLA-4 antagonistcan be any of the specific such CTLA-4 antagonists described in greaterdetail in below. In any of the embodiments, the isolated humanizedantibody that selectively binds and activates iNKT cells can be any ofthe humanized antibodies that selectively bind and activate iNKT cellsdescribed in greater detail below. In any of the embodiments, theisolated humanized antibody that selectively binds and activates iNKTcells can be NKTT320.

In any of the foregoing embodiments, the compositions and treatments maybe with or without CTLA4 antagonists, PD-1 antagonists, or both CTLA4and PD-1 antagonists.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the cytokine profile induced by NKT14m and by α-GalCer inhuman iNKT cells.

FIG. 2 shows the chemokine profile induced by NKT14m and by α-GalCer inhuman iNKT cells.

FIG. 3 shows the effects on several parameters after six weeks dosingwith α-GalCer versus NKT14m.

FIG. 4 is a graph showing the effects on survival on mice having a tumorimplanted and established and then treated with NKT14m, CTLA4, PDL1/2single antibodies, PDL1/2 antibodies+NKT14m combination antibodies andCTLA4+NKT14m combination antibodies.

FIG. 5 is a graph showing the effects on weight in mice having a tumorimplanted and then treated with NKT14m and CTLA4 single antibodies, andCTLA4+NKT14m combination antibodies.

DETAILED DESCRIPTION

The following detailed description is made by way of illustration ofcertain aspects of the invention. It is to be understood that otheraspects are contemplated and may be made without departing from thescope or spirit of the present disclosure. The following detaileddescription, therefore, is not to be taken in a limiting sense.Scientific and technical terms used herein have meanings commonly usedin the art unless otherwise specified. The definitions provided hereinare to facilitate understanding of certain terms used frequently hereinand are not meant to limit the scope of the present disclosure. Thesingular forms “a”, “an”, and “the” encompass the plural, unless thecontent clearly dictates otherwise. The term “or” is generally employedin its sense including “and/or” unless the content clearly dictatesotherwise.

Antibody that Activates iNKT Cells.

The invention involves use of an antibody that activates iNKT cells.Such an “Activating Antibody” is one that, when it binds to an iNKT cellin vivo, results in stimulating the iNKT cell to produce interferongamma, IL 4, IL10, or IL 13 versus blocking the activity of or depletingthe iNKT cell. Blocking the activity means that when the antibody bindsto an iNKT cell in vivo, it results in preventing or lessening theability of the iNKT cell to produce interferon gamma, IL 4, IL10, and/orIL 13. Such blocking antibodies include antibodies that result indepletion of iNKT cells when the antibody binds to an iNKT cell in vivo.Activating Antibodies typically have an Fc portion that does not bindFcγRI and C1q. In one embodiment, the Fc portion of the ActivatingAntibody does not bind FcγRI, C1q, or FcγRIII Antibodies with suchfunctionality, in general, are known. There are native antibodies, suchas antibodies with an IgG4 Fc region. There also are antibodies with Fcportions genetically or chemically altered to eliminate the Antibodydependent cell cytotoxicity (ADCC) and/or complement dependentcytotoxicity (CDC) functionality.

IgG4 has been used as an Activating Antibody, as it has a low affinityfor FcgammaRIγ. IgG4 can be modified to decrease its Fc effectorfunctions, making it even more suitable as an Activating Antibody. Forexample, an Fc region modified by two single residue substitutions isdescribed in “Elimination of Fc Receptor-dependent Effector Function ofa Modified IgG Monoclonal Antibody to Human CD4”, Truneh et al., TheJournal of Immunology, 1925-1933, 2000. Changes to the glycosylation ofthe Fc region also have been made to improve antibody basedtherapeutics. See for example, Glycosylation as a strategy to improveantibody-based therapeutics”, Jefferis, R., Nature Reviews, Vol 8, March2009, 226-234. All such activating modifications as described above arewithin the scope of the present invention.

In an embodiment, the antibody binds selectively the epitope defined bySEQ ID No. 1. In other embodiments, the antibody binds selectively theepitope defined by both SEQ ID Nos. 1 and 2.

NKTT320 is a humanized mAb which specifically recognizes human iTCR. Itis a modified IgG4 mAb with two amino acid changes introduced in thehinge region, one designed to stabilize the IgG4 heavy chain dimerformation, and the 2nd to reduce residual FcγR binding capacity. NKTT320and numerous other iNKT Activating Antibodies are described in U.S.patent application publication number 2013/0136735, the entiredisclosure of which is incorporated herein by reference.

Programmed Death (PD-1) Antagonist.

A PD-1 antagonist is a molecule that blocks the interaction of PD-1 withits ligand(s) PD-L1 and/or PD-L2 and/or prevents PD-1 activation.Programmed cell death protein 1 also known as PD-1 is a 288 amino acidcell surface protein molecule that in humans is encoded by the PDCD1gene. PDCD1 has also been designated as CD279 (cluster ofdifferentiation 279). This gene encodes a cell surface membrane proteinof the immunoglobulin superfamily. PD-1 is expressed in pro-B cells andis thought to play a role in their differentiation. See Shinohara T,Taniwaki M, Ishida Y, Kawaichi M, Honjo T (March 1995). “Structure andchromosomal localization of the human PD-1 gene (PDCD1)”. Genomics 23(3): 704-6. doi:10.1006/geno.1994.1562. PMID 7851902.] PD-1 is a memberof the extended CD28/CTLA-4 family of T cell regulators. [Ishida Y,Agata Y, Shibahara K, Honjo T (November 1992). “Induced expression ofPD-1, a novel member of the immunoglobulin gene superfamily, uponprogrammed cell death”. EMBO J. 11 (11): 3887-95. PMC 556898. PMID1396582.] PD-1 is expressed on the surface of activated T cells, Bcells, and macrophages.

PD-1 has two ligands, PD-L1 and PD-L2, which are members of the B7family. PD-L1 protein is upregulated on macrophages and dendritic cells(DC) in response to LPS and GM-CSF treatment, and on T cells and B cellsupon TCR and B cell receptor signaling, whereas in resting mice, PD-L1mRNA can be detected in the heart, lung, thymus, spleen, and kidney.PD-L1 is expressed on almost all murine tumor cell lines, including PA1myeloma, P815 mastocytoma, and B16 melanoma upon treatment with IFN-γ.PD-L2 expression is more restricted and is expressed mainly by DCs and afew tumor lines.

PD-1 and its ligands may negatively regulate immune responses. Theinvention involves the surprising discovery that interfering with PD-1activity while simultaneously stimulating iNKT cells with and iNKT cellactivating antibody results in strong anti-tumor activity, includingeffective activity against established tumors.

PD-1 activity may be interfered with by antibodies that bind selectivelyto and block the activity of PD-1 or that bind selectively to andprevent binding of PD-L1 or PD-L2 to PD-1. The interaction between PD-1and its ligands PD-L1 and PD-L2 can also be blocked by molecules otherthan antibodies that bind PD-1, PD-L1 or PD-L2 and prevent binding ofthe target to its ligand. Such molecules can be small molecules or canbe peptide mimetics of PD-L1 and PD-L2 that bind PD-1 but do notactivate PD-1. PD-1 antagonists include those described in U.S.Publications 20130280265, 20130237580, 20130230514, 20130109843,20130108651, 20130017199, and 20120251537, 2011/0271358, EP 2170959B1,the entire disclosures of which are incorporated herein by reference.See also M. A. Curran, et al., Proc. Natl. Acad. Sci. USA 107, 4275(2010) and S. L. Topalian, et al., New Engl. J. Med. 366, 2443 (2012);J. R. Brahmer, et al., New Engl. J. Med. 366, 2455 (2012), anti-PD-1antibody BMS-936558 (under development by Bristol-Meyers Squibb, andalso known as MDX-1106 or ONO-4538), the anti-PD-1 antibody CT-011 orpidilizumab (under development by CureTech), the anti-PD-1 antibodyMK-3475 (under development by Merck, and also known as SCH 900475), theanti-PD-L1 antibody BMS-936559 (under development by Bristol-MeyersSquibb, and also known as MDX-1105), and the anti-PD-L1 antibodyMPDL3280A or RG7446 (under development by Genentech/Roche). Agents thatinterfere bind to the DNA or mRNA encoding PD-1 also can act as PD-1inhibitors. Examples include a small inhibitory anti-PD-1 RNAi, a smallinhibitory anti-PD-L1 RNA, a small inhibitory anti-PD-L2 RNAi, ananti-PD-1 antisense RNA, an anti-PD-L1 antisense RNA, an anti-PD-L2antisense RNA, a dominant negative PD-1 protein, a dominant negativePD-L1 protein, or a dominant negative PD-L2 protein. PDL-2 fusionprotein AMP-224 (co-developed by Glaxo Smith Kline and Amplimmune) isbelieved to bind to and block PD-1.

CTLA-4 Antagonist.

CTLA-4 activity may be blocked by molecules that bind selectively to andblock the activity of CTLA-4 or that bind selectively to itscounter-receptors, e.g., CD80, CD86, etc. and block activity of CTLA-4.Blocking means inhibit or prevent the transmission of an inhibitorysignal via CTLA-4.

CTLA4 antagonists include, for example, inhibitory antibodies directedto CD80, CD86, and/or CTLA4; small molecule inhibitors of CD80, CD86,and CTLA4; antisense molecules directed against CD80, CD86, and/orCTLA4; adnectins directed against CD80, CD86, and/or CTLA4; and RNAiinhibitors (both single and double stranded) of CD80, CD86, and/orCTLA4.

Suitable anti-CTLA4 antibodies include humanized anti-CTLA4 antibodies,such as MDX-010 (ipilimumab), tremelimumab, and the antibodies disclosedin PCT Publication No. WO 2001/014424, PCT Publication No. WO2004/035607, U.S. Publication No. 2005/0201994, European Patent No. EP1212422 B1, U.S. Pat. Nos. 5,811,097, 5,855,887, 6,051,227, 6,984,720,7,034,121, 8,475,790, U.S. Publication Nos. 2002/0039581 and2002/086014, the entire disclosures of which are incorporated herein byreference. Other anti-CTLA-4 antibodies that can be used in a method ofthe present invention include, for example, those disclosed Hurwitz etal., Proc. Natl. Acad. Sci. USA, 95(17):10067-10071 (1998); Camacho etal., J. Clin. Oncology, 22(145): Abstract No. 2505 (2004) (antibodyCP-675206); Mokyr et al., Cancer Res., 58:5301-5304 (1998), and Lipsonand Drake, Clin Cancer Res; 17(22) Nov. 15, 2011.

8318916, EP1212422B1

Isolated.

The antibodies and other therapeutic molecules used herein are isolated.Isolated means, in the context of an antibody or other biologic, theantibody or other biologic has been removed from its natural milieu orhas been altered from its natural state. As such, isolated does notnecessarily reflect the extent to which the molecule has been removedfrom its natural milieu or has been altered from its natural state.However, it will be understood that an antibody or other biologic thathas been purified to some degree and to an extent to which it can beused for its intended therapeutic purpose is “isolated”.

Antibody.

The methods herein employ antibodies. The term antibody is used in thebroadest sense and specifically includes, for example, single monoclonalantibodies, antibody compositions with polyepitopic specificity, singlechain antibodies, and antigen-binding fragments of antibodies. Anantibody may include an immunoglobulin constant domain from anyimmunoglobulin, such as IgG1, IgG2, IgG3, or IgG4 subtypes, IgA(including IgA1 and IgA2), IgE, IgD or IgM.

An antigen-binding fragment means a portion of an intact antibody thatbinds antigen. Examples of antibody fragments include Fab, Fab′, F(ab′).sub.2, and Fv fragments; diabodies; linear antibodies (Zapata etal., Protein Eng. 8 (10): 1057-1062 [1995]); and single-chain antibodymolecules. Fv is the minimum antibody fragment containing a completeantigen-recognition binding site. This region consists of a dimer of oneheavy- and one light-chain variable domain in tight, non-covalentassociation. In this configuration the three CDRs of each variabledomain interact to define an antigen-binding site on the surface of theV.sub.H-V.sub.L dimer. Collectively, the six CDRs confer antigen-bindingspecificity to the antibody. The Fab fragment also contains the constantdomain of the light chain and the first constant domain (CH1) of theheavy chain. Fab fragments differ from Fab′ fragments by the addition ofa few residues at the carboxy terminus of the heavy chain CH1 domainincluding one or more cysteines from the antibody hinge region.F(ab′).sub.2 antibody fragments originally were produced as pairs ofFab′ fragments which have hinge cysteines between them. Other chemicalcouplings of antibody fragments are also known. In importantembodiments, the antibody is a full length antibody (i.e., contains anFc region, which can be IgG4 for example).

The antibodies used herein are humanized. Humanized forms of non-human(e.g., murine) antibodies then are chimeric immunoglobulins (includingfull length immunoglobulins), immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab′)2, scFv or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromthe non-human immunoglobulin. Humanized antibodies typically includehuman immunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues that are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin (Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-327 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)).

A composite antibody is an antibody that contains sequence segments fromdifferent antibodies. Humanized antibodies can be formed of a compositeof overlapping human sequences, with one segment of the CDR found in onehuman sequence and another segment of the same CDR found in anotherhuman sequence, each of the two sequences having common sequences at anoverlapping region where the segments meet. In an embodiment, thecomposite human sequence is free of known T cell epitopes. Inembodiments, the composite human sequence does not elicit an immuneresponse in humans. In any of the embodiments, the subject can be humanand the antibody can be a humanized antibody. In any of the embodiments,the antibody can be a composite antibody. In any of the embodiments, thesubject can be human and the antibody can be a fully human antibody. Afully human antibody is an antibody consisting only of human amino acidsequences.

Further details respecting antibodies and general methods of makingantibodies can be found in U.S. patent application publication number2013/0136735, the entire disclosure of which is incorporated herein byreference.

The antibodies bind selectively their targets, such as iNKT cells, PD-1,PD-L1, PD-L2, CTLA-4 and CTLA-4 ligands. An antibody that bindsselectively its target cells means it has the ability to be used invitro or in vivo to bind to and distinguish such target bearing tissuefrom other tissue types of the species, including other closely relatedcell types (e.g., in the case iNKT cells, distinguishing iNKT cells fromother types of NKT cells, other lymphocyte types, and all other tissuetypes) under the conditions in which the antibody is used, such as underphysiologic conditions. In an embodiment, the antibody binds selectivelyhuman iNKT cells. In an embodiment, the antibody binds to the CDR3 loopof iNKT cells.

Established Cancer.

The therapies described herein include treatment of an existing or‘established’ cancer, that is, one that exists and is detectable in thesubject.

Infection.

The invention is expected to be useful in treating infections, includingestablished and even chronic infections, including viral infection,bacterial infection, fungal infection and parasitic infection. Thus, thedisclosed combination therapy is useful to treat viral infectionsincluding, but are not limited to, immunodeficiency (e.g., HIV),papilloma (e.g., HPV), herpes (e.g., HSV), encephalitis, influenza(e.g., human influenza virus A), hepatitis (e.g. HCV, HBV), and commoncold (e.g., human rhinovirus).

Non-viral infections treatable by the invention include, but are notlimited to, infections cause by microoganisms including, but not limitedto, Actinomyces, Anabaena, Bacillus, Bacteroides, Bdellovibrio,Bordetella, Borrelia, Campylobacter, Caulobacter, Chlamydia, Chlorobium,Chromatium, Clostridium, Corynebacterium, Cytophaga, Deinococcus,Escherichia, Francisella, Halobacterium, Heliobacter, Haemophilus,Hemophilus influenza type B (HIB), Hypnomicrobium, Legionella,Leptspirosis, Listeria, Meningococcus A, B and C, Methanobacterium,Micrococcus, Myobacterium, Mycoplasma, Myxococcus, Neisseria,Nitrobacter, Oscillatoria, Prochloron, Proteus, Pseudomonas,Phodospirillum, Rickettsia, Salmonella, Shigella, Spirillum,Spirochaeta, Staphylococcus, Streptococcus, Streptomyces, Sulfolobus,Thermoplasma, Thiobacillus, Treponema, Vibrio, Yersinia, Cryptococcusneoformans, Histoplasma sp. (such as Histoplasma capsulatum), Candidaalbicans, Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii,Rickettsia typhi, Leishmania, Mycoplasma pneumoniae, Chlamydialpsittaci, Chlamydial trachomatis, Plasmodium sp. (such as Plasmodiumfalciparum), Trypanosoma brucei, Entamoeba histolytica, Toxoplasmagondii, Trichomonas vaginalis and Schistosoma mansoni.

The treatment according to the invention may also be with or withoutconcurrent treatment with an antigen containing vaccine. Suitableantigens used in vaccines are well known in the art and will not belisted here.

Subject.

“Subject” means a mammal, such as a human, a nonhuman primate, a dog, acat, a sheep, a horse, a cow, a pig or a goat. In an importantembodiment, the mammal is a human.

Treatment.

“Treat”, “treating” and “treatment” encompass an action that occurswhile a subject is suffering from a condition which reduces the severityof the condition (or a symptom associated with the condition) or retardsor slows the progression of the condition (or a symptom associated withthe condition). This is therapeutic treatment. “Treat”, “treating” and“treatment” also encompasses an action that occurs before a subjectbegins to suffer from the condition (or a symptom associated with thecondition) and which inhibits the onset of or reduces the severity ofthe condition (or a symptom associated with the condition). This isprophylactic treatment.

Subjects are treated with effective amounts of the solutions of theinvention. An “effective amount” of a compound generally refers to anamount sufficient to elicit the desired biological response, i.e., treatthe condition. As will be appreciated by those of ordinary skill in thisart, the effective amount of a compound described herein may varydepending on such factors as the condition being treated, the mode ofadministration, and the age and health of the subject.

For therapeutic treatment, an effective amount is an amount sufficientto provide a therapeutic benefit in the treatment of a condition or toreduce or eliminate one or more symptoms associated with the condition.This may encompass an amount that improves overall therapy, reduces oravoids symptoms or causes of the condition, or enhances the therapeuticefficacy of another therapeutic agent.

For prophylactic treatment, an effective amount is an amount sufficientto prevent, delay the onset of, or reduce the severity of a condition,or one or more symptoms associated with the condition, or prevent itsrecurrence. This may encompass an amount that improves overallprophylaxis or enhances the prophylactic efficacy of anotherprophylactic agent.

In general, effective amounts are administered to enhance an immuneresponse in the subject. In connection with a specific disease orcondition, “enhance an immune response” means to halt the developmentof, inhibit the progression of, reverse the development of, or otherwisereduce or ameliorate one or more symptoms of the disease or condition,for example, one or more symptoms of cancer or infectious disease.

Subject.

A subject as used herein means a human.

Pharmaceutical Compositions.

Humanized antibodies, biologics and other molecules can be administeredfor the treatment of various disorders in the form of pharmaceuticalcompositions. Such compositions include the therapeutic(s) and one ormore other pharmaceutically acceptable components. See Remington'sPharmaceutical Science (15th ed., Mack Publishing Company, Easton, Pa.(1980)). The preferred form depends on the intended mode ofadministration and therapeutic application. The compositions can alsoinclude, depending on the formulation desired,pharmaceutically-acceptable, non-toxic carriers or diluents, which aredefined as vehicles commonly used to formulate pharmaceuticalcompositions for animal or human administration. The diluent is selectedso as not to adversely affect the biological activity of the antibody.Examples of such diluents are distilled water, physiologicalphosphate-buffered saline, Ringer's solutions, dextrose solution, andHank's solution. In addition, the pharmaceutical composition orformulation may also include other carriers or nontoxic, nontherapeutic,nonimmunogenic stabilizers and the like.

Pharmaceutical compositions can also include large, slowly metabolizedmacromolecules such as proteins, polysaccharides such as chitosan,polylactic acids, polyglycolic acids and copolymers (such as latexfunctionalized SEPHAROSE™ (GE Healthcare Bio-Sciences Ltd.), agarose,cellulose, and the like), polymeric amino acids, amino acid copolymers,and lipid aggregates (such as oil droplets or liposomes).

Pharmaceutical compositions may be injectable compositions. Injectablecompositions include solutions, suspensions, dispersions, and the like.Injectable solutions, suspensions, dispersions, and the like may beformulated according to techniques well-known in the art (see, forexample, Remington's Pharmaceutical Sciences, Chapter 43, 14th Ed., MackPublishing Co., Easton, Pa.), using suitable dispersing or wetting andsuspending agents, such as sterile oils, including synthetic mono- ordiglycerides, and fatty acids, including oleic acid.

Injectable compositions that include an antibody or other biologicuseful in the invention may be prepared in water, saline, isotonicsaline, phosphate-buffered saline, citrate-buffered saline, and the likeand may optionally mixed with a nontoxic surfactant. Dispersions mayalso be prepared in glycerol, liquid polyethylene, glycols, DNA,vegetable oils, triacetin, and the like and mixtures thereof. Underordinary conditions of storage and use, these preparations may contain apreservative to prevent the growth of microorganisms. Pharmaceuticaldosage forms suitable for injection or infusion include sterile, aqueoussolutions or dispersions or sterile powders comprising an activeingredient which powders are adapted for the extemporaneous preparationof sterile injectable or infusible solutions or dispersions. Preferably,the ultimate dosage form is a sterile fluid and stable under theconditions of manufacture and storage. A liquid carrier or vehicle ofthe solution, suspension or dispersion may be a solvent or liquiddispersion medium comprising, for example, water, ethanol, a polyol suchas glycerol, propylene glycol, or liquid polyethylene glycols and thelike, vegetable oils, nontoxic glyceryl esters, and suitable mixturesthereof. Proper fluidity of solutions, suspensions or dispersions may bemaintained, for example, by the formation of liposomes, by themaintenance of the desired particle size, in the case of dispersion, orby the use of nontoxic surfactants. The prevention of the action ofmicroorganisms can be accomplished by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. Isotonic agents such as sugars, buffers,or sodium chloride may be included. Prolonged absorption of theinjectable compositions can be brought about by the inclusion in thecomposition of agents delaying absorption—for example, aluminummonostearate hydrogels and gelatin. Solubility enhancers may be added.

Sterile injectable compositions may be prepared by incorporating thetherapeutic in the desired amount in the appropriate solvent withvarious other ingredients, e.g. as enumerated above, and followed bysterilization, as desired, by, for example filter sterilization. In thecase of sterile powders for the preparation of sterile injectablesolutions, methods of preparation include vacuum drying andfreeze-drying techniques, which yield a powder of the active ingredientplus any additional desired ingredient present in a previouslysterile-filtered solution. Any suitable sterilization process may beemployed, such as filter sterilization, e.g. 0.22 micron filter ornanofiltration, gamma or electron beam sterilization, or pulsed whitelight. Other suitable sterilization processes include UtiSter (PegasusBiologics, Irvine Calif.) and those described in, e.g., U.S. Pat. No.6,946,098 and U.S. Pat. No. 5,730,933.

In various embodiments, the final solution typically is adjusted to havea pH between about 4 and about 9, between about 5 and about 7, betweenabout 5.5 and about 6.5, or about 6. The pH of the composition may beadjusted with a pharmacologically acceptable acid, base or buffer.Hydrochloric acid is an example of a suitable acid, and sodium hydroxideis an example of a suitable base. The hydrochloric acid or sodiumhydroxide may be in any suitable form, such as a 1N solution

A resultant injectable solution preferably contains an amount of one ormore therapeutics effective to treat a disease. In various embodiments,a therapeutic such as an antibody is present in an injectablecomposition at a concentration between about 0.0001 mg/ml and about 50mg/ml. In various embodiments, an antibody is present in an injectablecomposition at a concentration between about 0.01 mg/mL and about 10mg/mL.

Biologics such as antibodies also may be administered via other modes ofadministration known in the art. Such modes of administration includeinhalation, ingestion and topical application. Oral administration isalso possible for therapeutics, although this form of administration ismore challenging for certain biologics such as antibodies.

Example 1

A murine specific antibody (NKT14m) was created that can bindspecifically to and activate murine iNKT cells. The specificity ofNKT14m—that is, its ability to distinguish iNKT cells from other cellsincluding other NKT cells, was demonstrated and shown to be similar tothe specificity in binding observed for NKTT320 which binds specificallyhuman iNKT cells (but not murine iNKT cells).

Example 2

Experiments then were conducted to determine whether NKT14m induces asimilar cytokine and chemokine profile as compared to the profileobserved in human iNKT cells activated by α-GalCer. As shown in FIGS. 1and 2, the cytokine and chemokine profile induced by NKT14m wasqualitatively similar to that produced by α-GalCer in human iNKT cells.

Example 3

Experiments then were conducted to determine whether NKT14m activationof iNKT cells caused anergy similar to that caused by α-GalCeractivation of iNKT cells. The effects on several parameters weremeasured six weeks post doing with α-GalCer versus NKT14m. Unexpectedly,it was discovered that NKT14m dosing did not cause long lasting anergysimilar to that caused by α-GalCer dosing. This suggests that antibodyactivation of iNKT cells might be a better therapeutic strategy thanα-GalCer iNKT cell activation. See FIG. 3.

Example 4

The efficacy of NKT14m alone and in combination with other immunetherapies was assessed in murine models of melanoma (B16F10) and renalcell carcinoma (RENCA).

An outline of the B17F10 Melanoma study is found in Table 1

TABLE 1 Melanoma Study Groups No Adm. Treatment Treatment Group animalsTreatment Dose Route start schedule 1 10 + 3 IgG2a Isotype control 100μg/mouse IV D 3 Q1Dx1 2 10 + 3 NKT-14m 100 μg/mouse IV D 3 Q1Dx1 3 10Anti-CTLA4 100 μg/mouse IP D 3 Q4Dx3 4 10 anti PD-L1/PD-L2 300 μg firstdose IP D 3 Q3Dx4 200 μg thereafter 5 10 Anti-CTLA4 100 μg/mouse IP D 3Q4Dx3 NKT-14m 100 μg/mouse IV Q1Dx1 6 10 anti PD-L1/PD-L2 300 μg firstdose IP D 3 Q3Dx4 200 μg thereafter NKT-14m 100 μg/mouse IV Q1Dx1 7 10Doxorubicin 12 mg/kg IV D 3 Q1Dx1

An outline of the RENCA study is found in Table 2

TABLE 2 RENCA Study Groups No Adm. Treatment Treatment Group animalsTreatment Dose Route start schedule 1 10 IgG2a Isotype control 50μg/mouse IV D 0 Q1Dx1* 2 10 NKT-14m 50 μg/mouse IV D 0 Q1Dx1* 3 10 + 3IgG2a Isotype control 50 μg/mouse IV D 8 Q1Dx1* 4 10 Anti-CTLA4 100μg/mouse IP D 8 Q4Dx3 5 10 + 3 NKT-14m 50 μg/mouse IV D 8 Q1Dx1* 6 10Anti-CTLA4 100 μg/mouse IP D 8 Q4Dx3 NKT-14m 50 μg/mouse IV Q1Dx1* 7 10Sorafenib 100 mg/kg/adm PO D 8 Q1Dx21

Results

Melanoma. In the melanoma survival study, the positive control groupDoxorubicin had a significant effect in overall survival (70% survival).The NKT14m, CTLA4, PDL1/2 single antibodies had survivals of 10-20%. Thecombination of PDL1/2 antibodies+NKT14m had a 40% survival, suggesting asynergistic effect. See FIG. 4.

The combination of CTLA4+NKT14m did not improve survival beyond thatseen for the individual antibodies. Interestingly, however, thecombination of PDL1/2 antibodies+NKT14m showed no effect in testing theeffects of the antibodies and their combinations on tumor weight.

RENCA. In the RENCA study, CTLA4 and NKT14m single antibody andcombination treatment was tested. (A Sorafenib treatment group was usedas a control) had a primary median tumor weight that was significantlyreduced compared to the isotype control group. In the therapeutic dosinggroups, the combination of NKT14m+CTLA4 antibodies had a reduction intumor median weight compared to either agent administered alone, againsuggesting a synergistic effect of the combination. See FIG. 5.

SUMMARY

Overall the results suggest that, in established tumors, NKT14m incombination with other immune modulators can mediate an anti-tumoreffect.

1. A method for treating a human subject having a cancer or aninfection, comprising administering to the human subject an effectiveamount of (a) an isolated humanized antibody that selectively binds andactivates iNKT cells, and (b) an isolated Programmed Death (PD-1)antagonist, wherein the isolated humanized antibody and the isolatedProgrammed Death (PD-1) antagonist are administered in amounts effectiveto treat the cancer or the infection.
 2. (canceled)
 3. The method ofclaim 1, wherein the isolated Programmed Death (PD-1) antagonist is ahumanized antibody that selectively binds PD-1, PD-L1 or PD-L2. 4-5.(canceled)
 6. The method of claim 1, wherein the subject has cancer andthe isolated humanized antibody and the isolated Programmed Death (PD-1)antagonist are administered in amounts effective to treat the cancer. 7.The method of claim 6, wherein the cancer is melanoma, squamous cellcarcinoma, basal cell carcinoma, breast cancer, head and neck carcinoma,thyroid carcinoma, soft tissue sarcoma, bone sarcoma, testicular cancer,prostatic cancer, ovarian cancer, bladder cancer, skin cancer, braincancer, angiosarcoma, hemangiosarcoma, mast cell tumor, primary hepaticcancer, lung cancer, pancreatic cancer, gastrointestinal cancer, renalcell carcinoma, hematopoietic neoplasia, or a metastatic cancer thereof.8-9. (canceled)
 10. The method of claim 1, wherein the isolatedhumanized antibody that selectively binds and activates iNKT cellscomprises NKTT320.
 11. A pharmaceutical composition comprising aneffective amount of (a) an isolated humanized antibody that selectivelybinds and activates iNKT cells, and (b) an isolated Programmed Death(PD-1) antagonist.
 12. (canceled)
 13. The pharmaceutical composition ofclaim 11, wherein the isolated Programmed Death (PD-1) antagonist is ahumanized antibody that selectively binds PD-1 PD-L1 or PD-L2. 14-15.(canceled)
 16. The pharmaceutical composition of claim 11, wherein theisolated humanized antibody that selectively binds and activates iNKTcells comprises NKTT320.
 17. (canceled)
 18. A kit comprising a packagecontaining (a) an isolated humanized antibody that selectively binds andactivates iNKT cells, and (b) an isolated Programmed Death (PD-1)antagonist. 19-20. (canceled)
 21. A method for treating a human subjecthaving a cancer or an infection, comprising administering to the humansubject an effective amount of (a) an isolated humanized antibody thatselectively binds and activates iNKT cells, and (b) an isolated CTLA-4antagonist, wherein the isolated humanized antibody and the isolatedCTLA-4 antagonist are administered in amounts effective to treat thecancer or the infection.
 22. (canceled)
 23. The method of claim 21,wherein the isolated CTLA-4 antagonist is a humanized antibody thatselectively binds CTLA-4.
 24. (canceled)
 25. The method of claim 21,further comprising administering an isolated Programmed Death (PD-1)antagonist.
 26. The method of claim 21, wherein the subject has cancerand the isolated humanized antibody and the isolated CTLA-4 antagonistare administered in amounts effective to treat the cancer.
 27. Themethod of claim 26, wherein the cancer is melanoma, squamous cellcarcinoma, basal cell carcinoma, breast cancer, head and neck carcinoma,thyroid carcinoma, soft tissue sarcoma, bone sarcoma, testicular cancer,prostatic cancer, ovarian cancer, bladder cancer, skin cancer, braincancer, angiosarcoma, hemangiosarcoma, mast cell tumor, primary hepaticcancer, lung cancer, pancreatic cancer, gastrointestinal cancer, renalcell carcinoma, hematopoietic neoplasia, or a metastatic cancer thereof.28-29. (canceled)
 30. The method of claim 21, wherein the isolatedhumanized antibody that selectively binds and activates iNKT cellscomprises NKTT320.
 31. A pharmaceutical composition comprising aneffective amount of (a) an isolated humanized antibody that selectivelybinds and activates iNKT cells, and (b) an isolated CTLA-4 antagonist.32. (canceled)
 33. The pharmaceutical composition of claim 31, whereinthe isolated CTLA-4 antagonist is a humanized antibody that selectivelybinds CTLA-4. 34-35. (canceled)
 36. The pharmaceutical composition ofclaim 31, wherein the isolated humanized antibody that selectively bindsand activates iNKT cells comprises NKTT320. 37-40. (canceled)